Intracellular distribution of lipophilic fluorescent probes in mammalian cells

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and $\text{2-p-}\text{toluidinyl-6-naphthalene}$ sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Fluorescence spectroscopic studies of Huntington fibroblast membranes.

Using fluorescence spectroscopic methods, we compared the membrane properties of intact fibroblasts from both normal subjects and patients with Huntington disease (HD). Cells were stained with various fluorophores, including 1-anilino-8-naphthalene sulfonic acid (ANS), 2-toluidinyl-6-naphthalene sulfonic acid (TNS), 1,6-diphenyl-1,3,5-hexatriene (DPH), and 6-lauroyl-2-(dimethylamino)-naphthalene (LAURDAN). Using these labeled cells, we measured fluorescence yields and emission maxima (ANS, TNS, and LAURDAN), polarizations (TNS, DPH, and LAURDAN), lifetimes (TNS), and differential polarized lifetimes (DPH). In each instance, comparisons were made between cells from normal and from HD individuals. These cultures were controlled for passage number in culture and for age of donor. We found no significant differences between the HD and the control fibroblasts in experiments using the above-mentioned probes and spectroscopic parameters.     

Comparative study on fluorescent probes distributed in human erythrocytes and platelets

Important cellular functions, such as rheological properties of cells are presumably related to the membrane lipid fluidity which may be approached by the use of fluorescence polarization method. However, biological membranes represent very heterogeneous media and the knowledge of the fluidity of membrane compartments requires the use of different probes. Two fluorescent probes, DPH and its cationic derivative, TMA-DPH, have been employed to probe the lipid fluidity of human platelets and red cell membranes. The results show that the informations given by DPH and TMA-DPH can present important differences, suggesting that DPH and TMA-DPH are localized in different regions of cell membranes. In an attempt to investigate relations between lipid fluidity and rheological properties of red cells, the behavior of probes was studied in a "Couette" viscometer with a device for studying the emissive properties of probes when red cell membranes are under shear conditions.     

Characterization of the core and surface of human plasma lipoproteins. A study based on the use of five fluorophores.

The physical properties of the core and the surface of five classes of human plasma lipoproteins were investigated using five fluorescent probes. The location of the fluorescence probes in the lipoprotein assembly was determined using collisional quenching and resonance energy transfer. The fluorophores monitor different regions of the lipoproteins, as shown by fluorescence quenching. Diphenylhexatriene (DPH) and methyl trans-parinaric acid (MTPA), which are apolar molecules, are localized mainly in the lipoprotein core. Their distribution into the surface is dependent upon the volume ratio of the hydrophobic part of the envelope and the core. The polar fluorophores, trimethylaminodiphenylhexatriene (TMADPH), hydroxycoumarin (HC) and trans-parinaric acid (TPA) are anchored in the glycerol skeleton region of the surface monolayer with the fluorophore group of HC in the headgroup region of the phospholipids. We determined the temperature-dependent steady-state fluorescence anisotropy (r) of these fluorophores in the four major classes of lipoproteins: VLDL, LDL, HDL2, HDL3 and in abnormal HDL from abetalipoproteinemia patients (HDLab). The hydrophobic probes, DPH and MTPA, reported the r values in the lipoproteins in the following order: LDL greater than HDL2 greater than HDL3 much greater than VLDL. This order correlates with the triglyceride-to-cholesterol ester (TG/CE) ratio in the core of lipoproteins. The polar probes HC, TPA and TMADPH reported the r value in a different order: HDL2, HDL3 greater than or equal to LDL much greater than VLDL. This is compatible with the decreasing order of the protein to lipid ratio in the envelope of these lipoproteins. HDLab was investigated by three fluorescent probes: DPH, TMADPH and HC. The anisotropy of DPH in HDLab was larger than that of either HDL2 or HDL3 in normal donors, probably due to the smaller TG/CE ratio in HDLab. The lower r values reported by HC and TMADPH for HDLab are not fully understood and may be related to other factors such as acyl chains composition. The characterization of lipoproteins by fluorescence depolarization using probes of known location in the lipoprotein assembly is very sensitive and may be used to report deviation from the norm.     

Fluorescence of squid axon membrane labelled with hydrophobic probes

Summary Extrinsic fluorescence changes in squid giant axons were examined under a variety of experimental conditions using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and other fluorescent probes. Measurements of the degree of polarization of the fluorescent light (with the axis of the polarizer parallel to the longitudinal axis of the axon) indicated that the class of the TNS molecules in the axon membrane which participate in production of fluorescence signals have a definite orientation with their absorption and emission oscillators directed parallel to the long axis of the axon. Rectangular depolarizing voltage pulses produced a transient decrease in the fluorescent intensity, of which the early component is correlated tentatively with the rise in the membrane conductance. In response to hyperpolarizing pulses, there was an increase in fluorescence intensity which may be explained in terms of increased incorporation of TNS into the ordered structure in the membrane. Hyperpolarizing responses in KCl depolarized axons were accompanied by a change in fluorescent intensity. Tetrodotoxin appeared to suppress the initial component of the fluorescence signal produced by depolarizing clamping pulses. The technique for detecting these fluorescence changes and the physico-chemical properties of TNS are described in some detail.     

Localization of the lipid probe 1,6-diphenyl-1,3,5 hexatriene (DPH) in intact cells by fluorescence microscopy

The lipophilic fluorescent probe DPH, generally used to determine the microviscosity of membrane lipids, has been visualized in intact cells by fluorescence microscopy. All lipid material of the cells, including cytoplasmic lipid droplets, was found to be labelled with DPH. The fluorescent signal from inside the cells contributes to a large extent to the total cell fluorescence. The results indicate that fluorescence polarization data obtained from intact cells, using DPH as probe, give information on the total lipid material of the cells rather than exclusive information on microviscosity and fluidity of plasma membranes of these cells, as has been repeatedly suggested.     

Fluorescent Probes DPH,TMA-DPH and C17-HC Induce Erythrocyte Exovesiculation

An experimental approach has been developed to study human erythrocyte vesiculation, using the fluorescent probes diphenylhexatriene (DPH), trimethylamino-diphenylhexatriene (TMA-DPH) and heptadecyl-hydroxycoumarin (C17-HC). Acetylcholinesterase (AChE) enzyme activity measurements confirmed the presence of exovesicles released from erythrocyte membranes labeled with DPH, TMA-DPH or C17-HC. The fluorescence intensity and anisotropy values obtained showed that the amphiphilic probes TMA-DPH and C17-HC are preferentially incorporated in the exovesicles (when compared with DPH). There is a significant decrease of the cholesterol content of the exovesicle suspensions with time, independently of the fluorescence probe used, reaching undetectable cholesterol levels for the samples incubated for 48 hr. The ratios between the concentration of cholesterol released in the exovesicles after 1 hr incubation with DPH, TMA-DPH or C17-HC and the probe concentration used in the incubation were 84.7, 3.82 and 0.074, respectively. The size of the released vesicles was evaluated by dynamic light scattering spectroscopy. Some hypotheses are proposed that could explain the resemblance and differences between the results obtained for erythrocytes labeled with each probe, considering the present knowledge of membrane vesiculation mechanisms, lipid microdomains (rafts), erythrocyte membrane phospholipid asymmetry and AChE inhibition by TMA-DPH and C17-HC. This work demonstrates that the fluorescent probes DPH, TMA-DPH and C17-HC induce rapid erythrocyte exovesiculation; their use can lead to new methodologies for the study of this still poorly understood mechanism.     

Comparisons of steady-state anisotropy of the plasma membrane of living cells with different probes

We have used an extended Perrin equation which was in agreement with literature data for steady-state anisotropy (rSS) for a wide variety of artificial and isolated biological membranes labeled with various probes (Van der Meer et al. (1986) Biochim. Biophys. Acta 854, 38-44 to obtain the static component (r infinity) for the intact plasma membranes of living cells. We show that lipid structural order parameters can be obtained for DPH and TMA-DPH in the plasma membranes of intact cells. We have examined the relationship between 'fractional limiting hindered anisotropy', r infinity/r0, which is related to the lipid structural order parameter, of DPH, TMA-DPH, DPHpPC, and a series of depth-dependent probes (n-(9-anthroyloxy) fatty acids, with n = 2-16), using data from 19 cell types. There was a linear relationship between r infinity/r0 values of DPH and TMA-DPH, but the relationship between either of these probes was non-linear with respect to DPHpPC or the series of fatty acid probes. The relationship between r infinity/r0 values of DPHpPC and the series of fatty acid probes was linear, suggesting that they not only undergo similar motions in the membrane, but also experience similar types of restriction to motion, a type which is different from that experienced by DPH and TMA-DPH. We show that for the plasma membranes of living cells, 'second degree' order parameters can be estimated for DPH and TMA-DPH, and propose that the parameter r infinity/r0, or the 'fractional limiting hindered anisotropy', analogous to a 'first degree' order parameter, can be estimated for DPHpPC and the depth-dependent fatty acid probes to evaluate the density of membrane packing.     

Reactions of fluorescent probes with normal and chemically modified myelin.

The fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) bind to highly purified myelin membranes obtained from bovine brain white matter. Binding of the dyes was markedly increased by environmental conditions which reduce the negative surface potential of the membrane, i.e., cations (La-3+ is greater than Ca-2+ is greater than Na-+,K-+), H-+, local anesthetics, and the antibiotic polymyxin B. Chemical alteration of accessible membrane charged groups affected dye binding in a manner consistent with the hypothesis that such binding is primarily dependent upon the membrane surface potential. Thus, binding was increased by blocking of carboxyl groups via carbodiimide activation and subsequent coupling with neutral amino acid esters, and even more so with a basic amino acid ester (e.g., arginine methyl ester). Dye binding was reduced by succinylation of amino groups, and by hydrolysis of choline and ethanolamine head groups of phospho- and sphingolipids by phospholipase C. Phospholipase C treatment of myelin, or sphingomyelin vesicles, reduced or abolished the augmentation of ANS and TNS binding due to cations, local anesthetics, or polymyxin B. Energy transfer from myelin tryptophan residues to bound ANS occurs, but with low efficiency. Oxidation of membrane tryptophan residues with N-bromosuccinimide, or alkylation with 2-hydroxy (or methoxy)-5-nitrobenzyl bromide, markedly reduced intrinsic membrane fluorescence and energy transfer to bound ANS, but did not significantly affect dye binding or the quantum yield of ANS fluorescence when excitation was at 380nm. Proteolytic digestion removed 6-30% of myelin protein, depending upon the enzyme used, but had no effect on fluorescent dye binding. It is concluded that the binding of the anionic fluorescent probes ANS and TNS to myelin is primarily a function of the membrane surface charge density and net surface potential, as is the case with other biological membranes. Conclusions about the degree of dye binding to membrane lipids or membrane proteins cannot be drawn unless additional studies are carried out on isolated water soluble membrane proteins.     

Differences in the surface membranes and water content between the vegetative cells and spores of Bacillus subtilis

Bacillus subtilis forms both vegetative cells and spores. The fluidity of the membranes in these forms was measured by using fluorescent anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH). The spores were more rigid than the vegetative cells, suggesting that the structure of the spores and vegetative cells was different. This difference was thought to be due to the structure of the cell membranes. The anisotrophy of DPH in the cell membranes of spores gave higher values at all temperatures. The anisotrophy of DPH in the cell membranes of vegetative cells was lower than that of the spores and the value depended upon the temperature. Time Domain Reflectometry (TDR) was used to measure the quantities of bound and free water in the vegetative cells and spores. The spores were dehydrated, and the amount of bound and free water in the spores was about two‐thirds of the levels in the vegetative cells. The spores have fewer sugars molecules on their cell surface membranes, but contained as much sugars within the cell. Almost 100 per cent of the vegetative cells wee absorbed toward chitin, but the spores were not absorbed toward it at all. It was felt that the surface membrane of the vegetative cell had a high mobility because it was sugar‐rich, while the surface membrane of the spore showed a lower mobility because there are fewer sugars on the outer membrane. The spores survive in high temperatures because the surface membrane of the spore is tight and has relatively few sugars. Dehydration causes the rigidity of the spores. On the other hand, the vegetative cells are sugar‐ and water‐rich, which makes them more fluid. The difference between the vegetative cells and spores is the glycosylation of their surface membranes. Copyright © 1999 John Wiley & Sons, Ltd.     

Membrane effects of retinoids: Possible correlation with toxicity

The mechanism of toxicity of vitamin A and other retinoids is unknown. One theory holds that these compounds disrupt cellular function by acting on membranes as detergents would. Using rat erythrocyte ghosts labeled with fluorescent probes, we studied the changes in fluorescence polarization effected by detergents and retinoids. Fluorescence lifetime measurements allowed us to calculate apparent microviscosities. Sodium dodecyl sulfate, Triton X-100, and lysophosphatidylcholine seemed to cause a structural transition in the membrane where the apparent microviscosity was reduced by half at a detergent concentration of approximately 10^?3m; the same transition point was reported by 1,6-diphenylhexatriene or perylene. In contrast, retinoic acid reduced membrane microviscosity by half at a concentration of only 10^?5m or lower. Furthermore, the results obtained with DPH were very different from those obtained with perylene as the probe. The effect of retinoic acid was prevented by d-α-tocopherol acetate. Other retinoids were also effective in reducing membrane microviscosity, and the order of their effectiveness exactly paralleled the order of their toxicities, as determined by others using bioassays. These results suggest that retinoid toxicity may indeed be related to a membrane effect, but the action of retinoids on membranes is unlike that of simple detergents.     

Fluorescence polarization of six membrane probes in embryonal carcinoma cells after differentiation as measured on a FACS II cell sorter

Fluorescence polarization measurements on a FACS II cell sorter were compared with static measurements on a spectrofluorimeter using calibration solutions and Hoechst 33258-labeled cells. For the flow cytometric measurements on the FACS we used a pseudodepolarizer for normalization of the output of the two photomultipliers. The results showed that fluorescein and fluoresceinated bovine serum albumin (BSA) solutions gave identical values on both instruments. The mean value for fluorescence polarization of Hoechst 33258-labeled cells as measured on the FACS was the same as the value obtained with the spectrofluorimeter. Subsequently the fluorescence polarization of six different membrane probes was determined using differentiating embryonal carcinoma cells as a model system. Differentiation was induced by treatment of the cells with retinoic acid together with cyclic AMP. With diphenylhexatriene (DPH) the fluorescence polarization increased from I/I = 1.55 to 1.74 upon differentiation. With a charged analog of DPH (TMA-DPH) fluorescence polarization increased from I/I = 1.87 to 2.02. No appreciable changes in fluorescence polarization were observed in this cell system when anthroyloxysterate probes (12-AS, 9-AS, 6-AS, 2-AS) were used.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Dansyl chloride labeling of Pseudomonas aeruginosa treated with pyocin R1: change in permeability of the cell envelope

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, caused an increase in binding of fluorescent label, 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride), to sensitive cells. In pyocin R1-treated cells, cytoplasmic soluble proteins and crude ribosomes as well as cell envelopes were labeled by dansyl chloride. The amount of bound dye was proportional to the multiplicity of pyocin R1 and reached a maximal level at high multiplicity. In addition, pyocin R1 rapidly caused an increase in fluorescence intensity of the hydrophobic probes N-phenyl-1-naphthylamine, pyrene, and perylene, which were mixed with cells. These results show that pyocin R1 damages locally a cell envelope barrier to hydrophobic solutes and allows dyes to penetrate into the intracellular space across the barrier.     

TMA-DPH: a suitable fluorescence polarization probe for specific plasma membrane fluidity studies in intact living cells

Fluorescence intensity measurements and fluorescence microscopy data showed that TMA-DPH (trimethylammonium diphenylhexatriene), a cationic derivative of the fluorescence polarization probe DPH, has a considerably different behavior in L929 cultured cells than does its parent molecule. In contrast to DPH, it incorporates very rapidly in the plasma membranes of the treated cells, and remains specifically localized on the cell surface for at least 25 min. It can therefore be recommended for specific plasma membrane fluidity measurements in whole living cells. No relevant information about the localization of the probes could be obtained by other techniques used in parallel, namely: subcellular fractionation and fluorescence inhibition by trinitrobenzene sulfonate (TNBS).     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r_ss) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r_ss for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r_oo) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.     

Fluidity changes and chemical composition of lipoproteins in type IIa hyperlipoproteinemia

The chemical composition and the physical properties of lipoproteins (VLDL, LDL and HDL) were studied in two groups of patients: 14 healthy normolipidemic subjects and 15 type IIa familial hypercholesterolemic patients. The steady-state fluorescence anisotropy rs was estimated in lipoproteins by the fluorescence depolarization of two fluorescent probes: the DPH (1,6-diphenyl-1,3,5-hexatriene) and the TMA-DPH (1,4-trimethylammonium phenyl-6-1,3,5-hexatriene). A structured order parameter S was calculated from the DPH fluorescence anisotropy. The flow activation energies were calculated for LDL and HDL from both groups from the Arrhenius plots (log r DPH versus 1/T). By using TNBS (trinitrobenzene sulfonic acid) as a distance control quencher, the two probes were located in the outer shell of LDL. In HDL, TMA-DPH remained at the surface of the particles, while DPH was more deeply embedded in the lipid core. There was no difference in the physico-chemical properties of VLDL between the two groups studied. DPH fluorescence anisotropies were significantly increased in LDL and HDL from the hypercholesterolemic group compared to the control particles (P less than 0.05 and P less than 0.01, respectively). In LDL this modification of the fluorescence anisotropy can be related to a change in the lipid composition of particles. LDL from hypercholesterolemic patients contained significantly less triacylglycerol (P less than 0.01) and more cholesteryl ester (N.S.). Their cholesteryl ester to triacylglycerol ratio was significantly higher. In HDL, there was no difference in chemical composition between the two groups. The increase in DPH fluorescence anisotropy can be related to the presence of smaller particles in HDL from HC group. No difference was noted in the TMA-DPH fluorescence anisotropy at 37 degrees C in the LDL from the two groups. In contrast, TMA-DPH fluorescence anisotropy in HDL from hypercholesterolemic group was significantly higher than in control HDL. The flow activation energy of DPH was also significantly higher in both LDL and HDL from the hypercholesterolemic group than in control group particles. In both LDL and HDL from the control group, DPH fluorescence anisotropy was negatively correlated with TG/protein and TG/PL ratios and positively correlated with the CE/TG ratio. No correlation was observed between lipid composition and DPH fluorescence anisotropy values in hypercholesterolemic particles. The modification in fluidity parameters, especially the increase in the flow activation energies in LDL and HDL from hypercholesterolemic patients, could lead to a restriction of cholesterol movements in these particles. From a physiological point of view, this could represent a loss of functional capacity.     

Interaction of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene with biomembranes

The relationship between the conditions of membrane labelling by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and its fluorescence parameters was investigated. In the labelling solutions prepared by the usual method, the presence of DPH microcrystals was revealed which led to the lower resultant fluorescence anisotropy values. Lower labelling efficiency was observed with DPH solutions in tetrahydrofuran when compared with solutions in acetone. Modifications of the labelling procedure are proposed which give better reproducibility of the results. There modified method involves the preparation of a 2 X 10(-4) mol. 1(-1) DPH stock solution in acetone, a 100-fold dilution in an appropriate buffer, subsequent bubbling through with nitrogen for 30 min and mixing the resulting solution with cell/membrane suspension in a 1:1 (v/v) ratio. Changes in intensity, anisotropy and spectra of DPH fluorescence in the course of membrane labelling were studied. A two-stage model of the incorporation of DPH into membranes was proposed, according to which DPH molecules first quickly adhere to the membrane surface and then are slowly translocated to the apolar regions of the membrane.     

Influence of DPH on the structure and dynamics of a DPPC bilayer

We have conducted extensive molecular dynamics (MD) simulations together with differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) experiments to quantify the influence of free 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probes on the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer. Atomistic MD simulations show that in the membrane-water interface the influence of DPH is minor, whereas in the acyl-chain region DPH gives rise to major perturbations. In the latter case, DPH is found to influence a wide range of membrane properties, such as the packing and ordering of hydrocarbon tails and the lateral diffusion of lipid molecules. The effects are prominent but of local nature, i.e., the changes observed in the properties of lipid molecules are significant in the vicinity of DPH, but reduce rapidly as the distance from the probe increases. Long-range perturbations due to DPH are hence not expected. Detailed DSC and (2)H NMR measurements support this view. DSC shows only subtle perturbation to the cooperative behavior of the membrane system in the presence of DPH, and (2)H NMR shows that DPH gives rise to a slight increase in the lipid chain order, in agreement with MD simulations. Potential effects of other probes such as pyrene are briefly discussed.